Gary Franklin

The 5' flank of mouse H19 in an unusual chromatin conformation unidirectionally blocks enhancer-promoter communication.

BACKGROUNDnDuring mouse prenatal development, the neighbouring insulin-like growth factor II (Igf2) and H19 loci are expressed monoallelically from the paternal and maternal alleles, respectively. Identical spatiotemporal expression patterns and enhancer deletion experiments show that the Igf2 and H19 genes share a common set of enhancers. Deletion of a differentially methylated region in the 5 flank of the H19 gene partially relieves the repression of the maternal Igf2 and paternal H19 alleles in the soma. The mechanisms underlying the function of the 5 flank of the H19 gene are, however, unknown.nnnRESULTSnChromatin analysis showed that the 5 flank of the mouse H19 gene contains maternal-specific, multiple nuclease hypersensitive sites that map to linker regions between positioned nucleosomes. These features could be recapitulated in an episomal-based H19 minigene, which was propagated in human somatic cells. Although the 5 flank of the H19 promoter has no intrinsic silencer activity under these conditions, it unidirectionally extinguished promoter-enhancer communications in a position-dependent manner, without directly affecting the enhancer function.nnnCONCLUSIONSnThe unmethylated 5 flank of the H19 gene adopts an unusual and maternal-specific chromatin conformation in somatic cells and regulates enhancer-promoter communications, thereby providing an explanation for its role in manifesting the repressed state of the maternally inherited Igf2 allele.

Genomic imprinting and mammalian development

Genomic imprinting, which results in the mono-allelic expression of certain genes in a parent of origin-dependent manner, represents a specialized form of gene regulation which may be vitally important for mammalian development. The mechanisms which underlie imprinting and the molecular nature of the imprint itself remain elusive but most likely include epigenetic modifications of DNA, such as methylation and chromatin structure changes. It is clear, however, that many of the known imprinted genes play important developmental roles and that changes in the functional imprinting of some of these genes may have important pathological consequences, including placental abnormalities.

The sequential activation and repression of the human PDGF-B gene during chronic hypoxia reveals antagonistic roles for the depletion of oxygen and glucose.

Abstract Hypoxia and glucose deprivation, are important during many physiological and pathological processes. Cells respond to these stimuli by activating genes involved in the regulation of metabolism and angiogenesis. Platelet derived growth factor-B (PDGF-B) is involved in the regulation of angiogenesis and tumour progression and is induced by hypoxia. Most known hypoxia-induced genes are activated by the hypoxia inducible factor (HIF-1), via its binding to specific response elements. The mechanism of hypoxic induction and the effect of low glucose on PDGF-B expression have not been characterised. We show that PDGF-B exhibits a novel, biphasic regulation (induction, followed by repression below basal levels) in bladder carcinoma cells cultured under chronic hypoxia. We show that the repression observed after long-term hypoxia is due to glucose-depletion and that this can also abrogate short-term hypoxic induction. This is in contrast to the previous results showing that hypoxiahypogly-caemia elicit the same response. We also show that a putative hypoxia response element in the PDGF-B promoter is not sufficient for hypoxic induction, although it does function as a hypoxia independent enhancer element in hepatocellular carcinoma cells.

Evidence for hypoxia-induced neuronal-to-chromaffin metaplasia in neuroblastoma

We present evidence that in neuroblastoma, a pediatric malignancy of embryonal sympathetic origin, hypoxia, underlies a phenotypic switch from a primitive neuronal to a chromaffin cell type. This conclusion is based on morphological and molecular data on 116 clinical tumors and is supported by data on the phenotypic effects of hypoxia on neuroblastoma cell lines when studied in monolayer culture and as tumor xenografts. In the clinical material, extensive chromaffin features were seen in regions of chronic tumor hypoxia. This was the exclusive form of intratumoral maturation of stroma‐poor tumors and was also seen in stroma‐rich tumors, either exclusively or in combination with ganglion‐like cells. In neuroblastoma cell lines, hypoxia induced changes in gene expression associated with the chromaffin features observed in vivo. We therefore propose tumor hypoxia as a major cue determining phenotype in sympathetic tumors of neuroblastic origin. Because it appears to be reversible upon reoxygenation in monolayer culture, we suggest the term metaplasia for the phenomenon.

Cell-type-specific modulation of PDGF-B regulatory elements via viral enhancer competition: A caveat for the use of reference plasmids in transient transfection assays

The human platelet-derived growth factor-B (PDGF-B) gene has been shown to display a wide range of levels of mRNA transcription in a variety of cell types. Functional analyses of PDGF-B gene expression have begun to reveal intricate, cell-type-specific regulatory mechanisms involving multiple control elements. We have previously isolated and characterised several elements involved in the control of human PDGF-B gene expression in the JEG-3 choriocarcinoma cell line and in the breast cancer-derived cell line, ZR-75. Assessment of the positive or negative regulatory contributions of these elements was carried out using transient transfection assays. Such studies routinely require the inclusion of a reference plasmid in order to determine transfection efficiency. Here we show that competition for regulatory factors occurs in transfected cells between viral enhancers and elements regulating PDGF-B gene transcription. A frequently used reference plasmid which utilises the SV40 promoter and enhancer region to drive expression of a beta-galactosidase reporter gene was found to severely repress the activity of a co-transfected reporter construct containing the PDGF-B promoter and its intronic enhancer in JEG-3 cells. This competition was localised to the enhancer region of the SV40 regulatory sequences and surprisingly, the effect was reversed in ZR-75 cells; where increasing the amount of reference plasmid strongly stimulated the activity of the PDGF-B construct. These results imply that the same intronic region which functions equally well as an enhancer in two distinct cell-types, may operate in response to different transcription factor complements. Furthermore, this data demonstrates that the choice of reference plasmid and its quantitative use can be a crucial factor when examining putative regulatory elements by transient transfection methods.

A novel type of regulatory element is required for promoter-specific activity of the PDGF-B intronic enhancer region.

We have previously described a non-classical, promoter-specific enhancer for the human Platelet-Derived Growth Factor B (PDGF-B) gene. In JEG-3 choriocarcinoma cells the activity of the enhancer depends upon co-operation with a sequence (the Enhancer-Dependent cis Co-activator EDC element) within the promoter. The PDGF-B enhancer fails to activate heterologous promoters, indicating that promoter-specificity depends on an element within the enhancer that can recognise a target sequence within the promoter. Here we identify a sequence within the enhancer of the PDGF-B gene which directs activation of the PDGF-B promoter by distal cis-acting elements. This specifies the wild-type PDGF-B promoter as the target for the enhancer and has been designated the EDC specificity element (EDCse). The cell-type specific nature of this interaction is extended by the observation that the EDCse is also dispensable for enhancer activity in breast-cancer cells (ZR-75). Concomitant to this observation, JEG-3 and ZR-75 cells differ in the binding of nuclear factors to the EDCse. We discuss the relevance of the EDC/EDCse system in regulation of gene expression.

Mechanisms of transcriptional regulation.

The phenomenon of genomic imprinting, whatever its biological functions and evolutionary origins may be, ultimately manifests itself in the monoallelic expression of certain genes. The assumption has been, therefore, that the transcriptional regulatory systems of imprinted genes would directly or indirectly represent the principal focus of the imprinting process. As this chapter will discuss, however, our perceptions of gene regulation mechanisms are currently undergoing a radical re-evaluation and some of the emerging concepts may prove to be of importance to our understanding of how genomic imprinting functions.

THE MOLECULAR AND CELLULAR BIOLOGY OF GROWTH STIMULATORY PATHWAYS DURING HUMAN PLACENTAL DEVELOPMENT

The human placenta transiently expresses tumour-like properties, such as explosive cell proliferation, cell invasiveness and immune privilege. It is not clear whether or not all of these properties can be found coexpressed in single cytotrophoblastic cells, the main cell component of first trimester pregnancies (1–3). The attainment of the explosive, proliferative cell phenotype of cytotrophoblasts following blastocyst implantation is likely, however, to be a pivotal event in the generation of the “pseudomalignant” phenotype of the human placenta. At the end of the first trimester of pregnancy, the cytotrophoblasts permanently lose their proliferative potential and undergo accelerated rates of differentiation into the syncytiotrophoblasts (1–3). We have been interested, for a number of years, in understanding how the balance between trophoblast proliferation and differentiation is developmentally controlled. Given the “pseudomalignancy” of the early human placenta, the mechanisms underlying this process should be interesting not only for developmental biology, but also for tumor biology. This report presents an account of our research into these issues.